Compounds

ABSTRACT

The invention provides ratB polypeptides and DNA (RNA) encoding ratB polypetides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ratB polypeptides to screen for antibacteria, compounds.

RELATED APPLICATIONS

This application claims benefit of U.S. Ser. No. 60/037,857 filed Feb.7, 1997; U.S. Ser. No. 60/044,365 filed Apr. 28, 1997; U.S. Ser. No.60/044,366 filed Apr. 28, 1997 and U.S. Ser. No. 60/045,129 filed Apr.28, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the rat family, hereinafter referred to as “ratB”.

BACKGROUTND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

The frequency of Statphylococcus aureus infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Staphylococcus aureus strains which are resistant tosome or all of the standard antibiotics. This has created a demand forboth new anti-microbial agents and diagnostic tests for this organism.

Aminoacyl-tRNA synthetases (aaRS) catalyse the ligation of amino acidsto their cognate tRNA species in all cellular organisms. In general,each of the tventy amino acids that are incorporated into growingpolypeptide chains has a corresponding aaRS. However, it is now welldocumented that this is not universally true and that glutaminyl-tRNAsynthetase (QRS) activity is absent in all Gram-positive prokaryotesexamined, in some Gram-negative prokaryotes and in the plastids of some,and possibly all, eukalyotes. Despite the absence of glutaminyl-tRNAsynthetase activity, cells are clearly able to produce the Gln-tRNAGlnrequired for accurate protein synthesis. The mechanism by which this isachieved involves the fonnation of Glu-tRNAGln as an intermediate thatis produced by the misaminoacylation of tRNAGln by glutamyl-tRNAsynthetase (ERS). The ‘correct’ end product, Gln-tRNAGln, is formed fromGlu-tRNAGln by transfer of an amine group to the ligated glutamateresidue. This reaction is catalysed by a tRNA- and Mg2+/ATP-dependentamidotransferase. (RNA-dependent AmidoTransferase—RAT). Inhibition ofthis apparently ubiquitous reaction in Gram-positive organisms, and someGram-negative organisms, would effectively lead to Gln-tRNAGlnstarvation and to the synthesis of aberrant proteins and the consequentcessation of bacterial protein synthesis.

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown Bacillus subtilis PET112-like protein, encoded by part of the DNAsequence from GenBank deposition with accession number U49790 protein.

SUMNMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel ratB polypeptides by homology between the amino acidsequence set out in Table 1 [SEQ ID NO: 2] and a known amino acidsequence or sequences of other proteins such as Bacillus subtilisPET112-like protein, encoded by part of the DNA sequence from GenBankdeposition with accession number U49790 protein.

It is a further object of the invention to provide polynucleotides thatencode ratB polypeptides, particularly polynucleotides that encode thepolypeptide herein designated ratB.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding ratB polypcptides comprisingthe sequence set out in Table 1 [SEQ ID NO:1] which includes a fulllength gene, or a variant thereof.

In another particularly prefelTed embodiment of the invention there is anovel ratB protein from Staphylococcus aureus comprising the amino acidsequence of Table 1 [SEQ ID NO:2], or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Staphylococcus aureus WCUH 29 strain contained in the depositedstrain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding ratB, particularly Staphylococcus aureus ratB,including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of ratB and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStaphylococcus aureus referred to herein as ratB as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of ratB polypeptide encoded by naturally occurting alleles ofthe ratB gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned ratB polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing ratbexpression, treating disease, for example, disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, clarcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), assaying genetic variation, and administeringa ratB polypeptide or polynucleotide to an organism to raise animmunological response against a bacteria, especially a Staphylococcusaureus bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to ratB polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention there are providedantibodies against ratB polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypetideor polynuclcotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided ratB agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a ratB polynucleotide or a ratB polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Host cell” is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Compulational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto detemnine identity.

Preferred parameters for polypeptide sequence comparison include thefollowing:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioed parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include thefollowing:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genietics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

Preferred polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide having at least a 50,60, 70,80, 85, 90, 95, 97 or 100% identity to a polynucleotide referencesequence of SEQ ID NO:1, wherein said reference sequence may beidentical to the sequence of SEQ ID NO: 1 or may include up to a certaininteger number of nucleotide alterations as compared to the referencesequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by thenumerical percent of the respective percent identity and subtractingthat product from said total number of nuelcotides in SEQ ID NO:1, or:

 n _(n) ≦x _(n)−(x _(n) •y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, and y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integerproduct of x_(n) and y is rounded down to the nearest integer prior tosubtracting it from x_(n). Alterations of a polynucleotide sequenceencoding the polypeptide of SEQ ID NO:2 may create nonsense, mis-senseor frameshift mutations in this coding sequence and thereby alter thepolypeptide encoded by the polynucleotide following such alterations.

Preferred polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 50,60, 70, 80,85, 90, 95, 97 or 100% identity to a polypeptide reference sequence ofSEQ ID NO:2, wherein said reference sequence may be identical to thesequence of SEQ ID NO: 2 or may include up to a certain integer numberof amino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carbxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the aminno acids inthe reference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the numerical percent of the respective percent identity andsubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) •y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, and y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integerproduct of x_(a) and y is rounded down to the nearest integer prior tosubtracting it from x_(a).

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypcptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a htiple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just ttvo examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpuiposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified fomis of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,fonrylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIFS, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synithesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel ratB polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel ratB of Staphylococcusaureus, which is related by amino acid sequence homology to Bacillussubtilis PET112-like protein, encoded by part of the DNA sequence fromGenBank deposition with accession number U49790 polypeptide. Theinvention relates especially to ratB having the nucleotide and aminoacid sequences set out in Table 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO:2] respectively, and to the ratB nucleotide sequences of the DNA in thedeposited straim and amino acid sequences encoded thereby.

TABLE 1 ratB Polynucleotide and Polypeptide Sequences (A) Sequences fromStaphylococcus aureus ratB polynucleotide sequence [SEQ ID NO:1]. 5′-ATGCATTTTGAAACAGTTATAGGACTTGAAGTTCACGTAGAGTTAAAAACGGACTCAAAAATGTTTTCTCCATCACCAGCGCATTTTGGAGCAGAACCTAACTCAAATACAAATGTTATCGACTTAGCATATCCAGGTGTCTTACCAGTTGTTAATAAGCGTGCAGTAGACTGGGCAATGCGTGCTGCAATGGCACTAAATATGGAAATCGCAACAGAATCTAAGTTTGACCGTAAGAACTATTTCTATCCAGATAATCCAAAAGCATATCAAATTTCTCAATTTGATCAACCAATTGGTGAAAATGGATATATCGATATCGAAGTCGACGGTGAAACAAAACGAATCGGTATTACTCGTCTTCACATGGAAGAAGATGCTGGTAAGTCAACACATAAAGGTGAGTATTCATTAGTTGACTTGAACCGTCAAGGTACACCGCTAATTGAAATCGTATCTGAACCAGATATTCGTTCACCTAAAGAAGCATATGCATATTTAGAAAAATTACGTTCAATTATTCAATACACTGGTGTATCAGACGTTAAGATGGAAGAGGGATCTTTACGTTGTGATGCTAACATCTCTTTGCGTCCATATGGTCAAGAAAAATTTGGTACTAAAGCCGAATTGAAAAACTTAAACTCATTTAACTATGTACGTAAAGGTTTAGAATATGAAGAAAAACGCCAAGAAGAAGAATTGTTAAATGGTGGAGAAATCGGACAAGAAACACGTCGATTTGATGAATCTACAGGTAAAACAATTTTAATGCGTGTTAAAGAAGGTTCTGATGATTACCGTTACTTCCCAGAGCCTGACATTGTACCTTTATATATTGATGATGCTTGGAAAGAGCGTGTTCGTCAGACAATTCCTGAATTACCAGATGAGCGTAAGGCTAAGTATGTAAATGAATTAGGTTTACCTGCATACGATGCACACGTATTAACATTGACTAAAGAAATGTCAGATTTCTTTGAATCAACAATTGAACACGGTGCAGATGTTAAATTAACATCTAACTGGTTAATGGGTGGCGTAAACGAATATTTAAATAAAAATCAAGTAGAATTATTAGATACTAAATTAACACCAGAAAATTTAGCAGGTATGATTAAACTTATCGAAGACGGAACAATGAGCAGTAAAATTGCGAAGAAAGTCTTCCCAGAGTTAGCAGCTAAAGGTGGTAATGCTAAACAGATTATGGAAGATAATGGCTTAGTTCAAATTTCTGATGAAGCAACACTTCTAAAATTTGTAAATGAAGCATTAGACAATAACGAACAATCAGTTGAAGATTACAAAAATGGTAAAGGCAAAGCTATGGGCTTCTTAGTTGGTCAAATTATGAAAGCGTCTAAAGGTCAAGCTAATCCACAATTAGTAAATCAACTATTAAAACAAGAATTAGATAAAAGA-3′ (B) ratB polypeptide sequence deduced fromthe polynucleotide sequence in this table [SEQ ID NO:2]. NH₂-MHFETVIGLEVHVELKTDSKMFSPSPAHFGAEPNSNTNVIDLAYPGVLPVVNKRAVDWAMRAAMALNMEIATESKFDRKNYFYPDNPKAYQISQFDQPIGENGYIDIEVDGETKRIGITRLHMEEDAGKSTHKGEYSLVDLNRQGTPLIEIVSEPDIRSPKEAYAYLEKLRSIIQYTGVSDVKMEEGSLRCDANISLRPYGQEKFGRKAELKNLNSFNYVRKGLEYEEKRQEEELLNGGEIGQETRRFDESTGKTILMRVKEGSDDYRYFPEPDIVPLYIDDAWKERVRQTIPELPDERKAKYVNELGLPAYDAHVLTLTKEMSDFFESTIEHGADVKLTSNWLMGGVNEYLNKNQVELLDTKLTPENLAGMIKLIEDGTMSSKIAKKVFPELAAKGGNAKQIMEDNGLVQISDEATLLKFVNEALDNNEQSVEDYKNGKGKAMGFLVGQIMKASKGQANPQLVNQLLKQELDKR-COOH (C)Polynucleotide sequence embodiments [SEQ ID NO:1].X-(R₁)_(n)-ATGCATTTTGAAACAGTTATAGGACTTGAAGTTCACGTAGAGTTAAAAACGGACTCAAAAATGTTTTCTCCATCACCAGCGCATTTTGGAGCAGAACCTAACTCAAATACAAATGTTATCGACTTAGCATATCCAGGTGTCTTACCAGTTGTTAATAAGCGTGCAGTAGACTGGGCAATGCGTGCTGCAATGGCACTAAATATGGAAATCGCAACAGAATCTAAGTTTGACCGTAAGAACTATTTCTATCCAGATAATCCAAAAGCATATCAAATTTCTCAATTTGATCAACCAATTGGTGAAAATGGATATATCGATATCGAAGTCGACGGTGAAACAAAACGAATCGGTATTACTCGTCTTCACATGGAAGAAGATGCTGGTAAGTCAACACATAAAGGTGAGTATTCATTAGTTGACTTGAACCGTCAAGGTACACCGCTAATTGAAATCGTATCTGAACCAGATATTCGTTCACCTAAAGAAGCATATGCATATTTAGAAAAATTACGTTCAATTATTCAATACACTGGTGTATCAGACGTTAAGATGGAAGAGGGATCTTTACGTTGTGATGCTAACATCTCTTTGCGTCCATATGGTCAAGAAAAATTTGGTACTAAAGCCGAATTGAAAAACTTAAACTCATTTAACTATGTACGTAAAGGTTTAGAATATGAAGAAAAACGCCAAGAAGAAGAATTGTTAAATGGTGGAGAAATCGGACAAGAAACACGTCGATTTGATGAATCTACAGGTAAAACAATTTTAATGCGTGTTAAAGAAGGTTCTGATGATTACCGTTACTTCCCAGAGCCTGACATTGTACCTTTATATATTGATGATGCTTGGAAAGAGCGTGTTCGTCAGACAATTCCTGAATTACCAGATGAGCGTAAGGCTAAGTATGTAAATGAATTAGGTTTACCTGCATACGATGCACACGTATTAACATTGACTAAAGAAATGTCAGATTTCTTTGAATCAACAATTGAACACGGTGCAGATGTTAAATTAACATCTAACTGGTTAATGGGTGGCGTAAACGAATATTTAAATAAAAATCAAGTAGAATTATTAGATACTAAATTAACACCAGAAAATTTAGCAGGTATGATTAAACTTATCGAAGACGGAACAATGAGCAGTAAAATTGCGAAGAAAGTCTTCCCAGAGTTAGCAGCTAAAGGTGGTAATGCTAAACAGATTATGGAAGATAATGGCTTAGTTCAAATTTCTGATGAAGCAACACTTCTAAAATTTGTAAATGAAGCATTAGACAATAACGAACAATCAGTTGAAGATTACAAAAATGGTAAAGGCAAAGCTATGGGCTTCTTAGTTGGTCAAATTATGAAAGCGTCTAAAGGTCAAGCTAATCCACAATTAGTAAATCAACTATTAAAACAAGAATTAGATAAAAGA-(R₂)_(n)-Y (D) Polypeptide sequenceembodiments [SEQ ID NO:2].X-(R₁)_(n)-MHFETVIGLEVHVELKTDSKMFSPSPAHFGAEPNSNTNVIDLAYPGVLPVVNKRAVDWAMRAAMALNMEIATESKFDRKNYFYPDNPKAYQISQFDQPIGENGYIDIEVDGETKRIGITRLHMEEDAGKSTHKGEYSLVDLNRQGTPLIEIVSEPDIRSPKEAYAYLEKLRSIIQYTGVSDVKMEEGSLRCDANISLRPYGQEKFGTKAELKNLNSFNYVRKGLEYEEKRQEEELLNGGEIGQETRRFDESTGKTILMRVKEGSDDYRYFPEPDIVPLYIDDAWKERVRQTIPELPDERKAKYVNELGLPAYDAHVLTLTKEMSDFFESTIEHGADVKLTSNWLMGGVNEYLNKNQVELLDTKLTPENLAGMIKLIEDGTMSSKIAKKVFPELAAKGGNAKQIMEDNGLVQISDEATLLKFVNEALDNNEQSVEDYKNGKGKAMGFLVGQIMKASKGQANPQLVNQLLKQELDKR-(R₂)_(n)-Y (E)Sequences from Staphylococcus aureus ratB polynucleotide ORF sequence[SEQ ID NO:3].5′-CTGAAAAACTTAAACTCATTTAACTATGTACGTAAAGGTTTAGAATATGAAGAAAAACGCCAAGAAGAAGAATTGTTAAATGGTGGAGAAATCGGACAAGAAACACGTCGATTTGATGAATCTACAGGTAAAACAATTTTAATGCGTGTTAAAGAAGGTTCTGATGATTACCGNTACTTCCCAGAGCCTGACATTGTACCTTTATATATTGATGATGCTTGGAAAGAGCGTGTTCGTCAGACAATTCCTGAATTACCAGATGAGCGTAAGGCTAAGTATGTAAATGAATTAGGTTTACTGCATACGATGCNCNCGTATTAACNTTGACTAAAGAAATGTCAGATTTCTTTGAATCAACAATTGGAACNAGGGTGCAGATTGTTAAATTTAACATCTAACTGGGTTAATGGGTGGCGTNAACGAATATTTAAATAAAAATCAANTAGAATTATTAGATACTAAATTAACACCAGAAAATTTAGCAGGTATGATTAAACTTATCGAAGACGGAACAATGAGCAGTAAAATTGCGAAGAAAGTCTTCCCAGAGTTAGGCAGCTAAAGGGTGGGTAATGCTAAACAGATTATGGGANGATAATGGCTNAGTTCAAATTTCTTGATGGAAGCAACAATCTTCTAAAATTGGGTANATGGAAGCATTAGACAAATAACGAACAATCNGTGGAAGATTACAAAAATGGTAAAGGCAAAGCTATGGGGCTTCTTAGTTGGTCAAATTATGAAAGCGTCTAAAGGTCAAGCTAATCCACAATTAGTAAATCAACTATTAAAACAAGAATTAGATAAAAGATAATTTANATCATCAAACTATGAAGATTTAAAAAATAAACCCTTGATTGCTGACTTAGATGCAATCGAGGGTTTATTTATATCTATAGAAGTCATATTACTTTTAACTTTATTCATTGNACATGTTAATGGTAAAAATATTAATTTTATTAATGCGTTAGCTTTAATTATATTAAGGCAAACTGTATAATAAAAAGGTATAAAACATTTGTGTATAAAGACAACATTATATTTACAACATCATTTTAAAGGTAAAATAGCATAACTGACGAAGTCTATATAATGAAGAACGGCAAAAAATGCTGAATAAATAACAAGCTTTGTACATATTGAGATAGTATTTGTTTAAGATACAAGTTGGTCTTTAACGATATTAAGAATGATGAAATAAGACTGAGCCTGGGTCATAAATTCAATGTCCTAGGCACTACAATGTTAATATTGGCAGTAGTTGACTGAAAGAAAATACGCTTGTAACAAGCTTNNNTCAATTCTAGTGGGGCCCCAACATAGAAGCTGACTTTCTGTCAGCTTACAATAATGTGCAAGTNGGGGTGGGGCCCCAAACAAAGAGAATTTCGAAAGGAAATTCTACAGACAATGCAAGTTGGGGTAGAACGAAATAAATTTTGTTAAATATTATTTCTGTCCCACTCCCTATTAGACGAAACAAAGATGAAGTCAAAATATATGAATTTTAAGTAGAAGGATAAGATATGAACAAACGTGCTAGAATCATTTATAACCCGACATCAGGTAAAGAGCTAATTTAAAAGAGAATTACCTGATGCCTTAATAAAATTAGAAAAAGCGGGATATGAAACGAGTGCATATGCAACCGAGAAAATAGGTGATGCCACACTTGAAGCAGAAAGAGCTATGCATGAAAATTATGATGTATTAATCGCTGCAGGTGGTGATGGAACATTAAATGAAGTAGTTAATGGTATCGCAGAAAAGCCTAATCGTCCTAAGCTAGGTGTCATTCCTATGGGTACTGTTAATGACTTTGGACGTGCATTGCATATACCTAATGACATCATGGGGGCACTTGATGTCATCATTGAAGGTCATTCTACTAAAGTAGATATTGGTAAAATGAATAATCGATACTTTATTAATTTAGCTGCAGGCGGACAATTGACGCAAGTCTCTTATGAAACACCGAGTAAATTGAAATCTATTGTTGGTCCATTTGCTTATTACATCAAAGGTTTCGAAATGTTACCTCAAATGAAAGCTGTAGATTTAAGAATTGAATATGATGGTAATGTTTNCCAAGGAGAAGCATTATTATTCTTTNTAGGCTTAACAAATCCAATGGCAGGATTCGAAAAATTAGTGCCCGGACGCTAAGTTAGATGACGGCTATTTCTACGTTNAATNTATAG-3′ (F) RatB polypeptidesequence deduced from the polynucleotide ORF sequence in this table [SEQID NO:4]. NH₂-MKNLNSFNYVRKGLEYEEKRQEEELLNGGEIGQETRRFDESTGKTILMRVKEGSDDYRYFPEPDIVPLYIDDAWKERVRQTIPELPDERKAKYVNELGLLHTMXXY-COOH

Deposited Materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, andreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

The deposited strain contains the full length ratB gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Puiposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Table 1[SEQ ID NO:2] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of ratB, and also those which have at least 70% identity to apolypeptide of Table 1 [SEQ ID NOS:2 and 4] or the relevant portion,preferably at least 80% identity to a polypeptide of Table 1 [SEQ IDNOS:2 and 4], and more preferably at least 90% similarity (morepreferably at least 90% identity) to a polypeptide of Table 1 [SEQ IDNOS:2 and 4] and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to a polypeptide of Table 1 [SEQID NOS:2 and 4] and also include portions of such polypeptides with suchportion of the polypeptide generally containing at least 30 amino acidsand more preferably at least 50 amino acids.

The invention also includes polypeptides of the formula set forth inTable 1 (D) [SEQ ID NO:2] wherein, at the amino terminus, X is hydrogen,and at the carboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is anyamino acid residue, and n is an integer between 1 and 1000. Any stretchof amino acid residues denoted by either R group, where R is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with ratB polypeptides fragments maybe “free-standing,” or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NOS:2 and 4], orof variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Staphylococcus aureus, arealso preferTed. Further preferred are fragments characterized bystructural or functional attributes such as fi-agments that comprisealpha-helix and alpha-helix foiiing regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of ratB, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

RatB polypeptides of the invention can interact with ratC and/or ratApolypeptides comprising a polyeptide sequence set forth in Table 2, toform ratB:ratA and ratC:ratB heterodimers or ratA:ratB:ratChetertrimers. Such heterotrimers and heterotrimers are useful in themethods of the invention, particularly vaccine and drug screeningmethods set forth herein.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the ratB polypeptide havinga deduced amino acid sequence of Table 1 [SEQ ID NOS:2 and 4] andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NOS:1 and 3], a polynucleotide of theinvention encoding ratB polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH29 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a sequence given in Table 1 [SEQ ID NOS:1 and 3],typically a library of clones of chromosomal DNA of Staphylococcusaureus WCUH 29 in E.coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 [SEQ ID NO:1] was discovered in a DNA library derivedfrom Staphylococcus aureus WCUH 29.

The DNA sequence set out in Table 1 [SEQ ID NOS:1] contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 [SEQ ID NOS:2] with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. The start codon of the DNA in Table 1 isnucleotide number 1 and last codon that encodes an amino acid is number1425, the stop codon being the next codon following this last codonencoding an amino acid.

RatB of the invention is structurally related to other proteins of therat family, as shown by the results of sequencing the DNA encoding ratBof the deposited strain. The protein exhibits greatest homology toBacillus subtilis PET112-like protein, encoded by part of the DNAsequence from the GenBank deposition with accession number U49790protein among known proteins. RatB of Table 1 [SEQ ID NO:2] has about93% identity over its entire length and about 96% similarity over itsentire length with the amino acid sequence of Bacillus subtilisPET112-like protein, encoded by part of the DNA sequence from theGenBank deposition with accession number U49790 polypeptide.

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in Table 1 [SEQ ID NO:1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro-protein sequence. The polynucleotidemay also contain non-coding sequences, including for example, but notlimited to non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences, termination signals, ribosome binding sites,sequences that stabilize mRNA, introns, polyadenylation sigals, andadditional coding sequence which encode additional amino acids. Forexample, a marker sequence that facilitates purification of the fusedpolypeptide can be encoded. In certain embodiments of the invention, themarker sequence is a hexa-histidine peptide, as provided in the pQEvector (Qiagen, Inc.) and described in Gentz el al., Proc. Natl. Acad.Sci., USA 86. 821-824 (1989), or an HA tag (Wilson et al., Cell 37: 767(1984). Polynucleotides of the invention also include, but are notlimited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A prefenred embodiment of the invention is the polynucleotide ofcomprising nucleotide 1 to 1425 set forth in SEQ ID NO:1 of Table 1which encodes the ratB polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C)[SEQ ID NO:1] wherein, at the 5′ end of the molecule, X ishydrogen, and at the 3′ end of the molecule, Y is hydrogen or a metal,R₁ and R₂ is any nucleic acid residue, and n is an integer between 1 and1000. Any stretch of nucleic acid residues denoted by either R group,where R is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus ratB havingthe amino acid sequence set out in Table 1 [SEQ ID NO:2]. The term alsoencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, intenruptedby integrated phage or an insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingratB variants, that have the amino acid sequence of ratB polypeptide ofTable 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, that do not alter the properties and activitiesof ratB.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding ratB polypeptide having an amino acid sequence set out in Table1 [SEQ ID NOS:2 and 4], and polynucleotides that are complementary tosuch polynucleotides. Altemnatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding ratB polypeptide ofthe deposited strain and polynucleotides complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthto the same are particularly preferred, and among these particularlypreferred polynucleotides, those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferredamong those with at least 95%, and among these those with at least 98%and at least 99% are particularly highly preferred, with at least 99%being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 [SEQ ID NO:1].

A further preferred embodiment of the invention provides apolynucleotide sequence comprising a ratB polynucleotide sequence andratC polynucleotide set forth in Table 2 [SEQ ID NO:3]. Anotherprefenred embodiment of the invention provides a polynucleotide sequencecomprising ratB polynucleotide sequence and ratA polynucleotide setforth in Table 2 [SEQ ID NO:5]. Yet another preferred embodiment of theinvention provides a polynucleotide sequence comprising the ratBpolynucleotide sequence, the ratA polynucleotide sequence set forth inTable 2 [SEQ ID NO:5] and the ratC set polynucleotide sequence forth inTable 2 [SEQ ID NO:3].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the tenns “stringent conditions” and “stringent hybridizationconditions” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH17.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in0.1×SSC at about 65° C. Hybridization and wash conditions are well knownand exemplified in Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularlyChapter 11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 or SEQ ID NO:3 under stringent hybridization conditions witha probe having the sequence of said polynucleotide sequence set forth inSEQ ID NO:1 or a fragment thereof, and isolating said DNA sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding ratB and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the ratB gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the ratB gene may be isolated byscreening using the known DNA sequence provided in SEQ ID NO: 1 tosynthesize an oligonucleotide probe. A labeled oligonucleotide having asequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the libraiy the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polyleptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses Such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECUIAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive confoimationi when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the ratB polynucleotides ofthe invention for use as diagnostic reagents. Detection of ratB in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also“individual(s)”), particularly mammals, and especially humans, infectedwith an organism comprising the ratB gene may be detected at the nucleicacid level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled ratB polynucleotide sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in the electrophloreticmobility of the DNA fragments in gels, with or without denaturingagents, or by direct DNA sequencing. See, e.g., Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and S I protectionor a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nuclcic acidencoding ratB can be used to identify and analyze mutations. Examples ofrepresentative primers are shown below in Table 2. The invention furtherprovides these primers with 1, 2, 3 or 4 nucleotides removed from the 5′and/or the 3′ end. These primers may be used for, among other things,amplifying ratB DNA isolated from a sample derived from an individual.The primers may be used to amplify the gene isolated from an infectedindividual such that the gene may then be subject to various techniquesfor elucidation of the DNA sequence. In this way, mutations in the DNAsequence may be detected and used to diagnose infection and to serotypeand/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, and most preferably disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyclitis), comprising deteinining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of Table 1 [SEQ ID NO: 1]. Increased or decreasedexpression of ratB polynucleotide can be measured using any on of themethods well known in the art for the quantation of polynucleotides,such as, for example, amplification, PCR, RT-PCR, RNase protection,Northem blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of ratB protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to deteimine levels of a ratBprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearincgfragments, analogues or cells to an animal, preferably a nonhuman, usingroutine protocols. For preparation of monoclonal antibodies, anytechnique known in the art that provides antibodies produced bycontinuous cell line cultures can be used. Examples include varioustechniques, such as those in Kohler, G. and Milstein, C., Nature 256.495-497 (1975); Kozbor el al., Immunology Today 4: 72 (1983); Cole etal., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfirm repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-ratB or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity clromatography.

Thus, among others, antibodies against ratB-polypeptide may be employedto treat infections, particularly bacterial infections and especiallydisease, such as, infections of the upper respiratory tract (c.g.,otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis),lower respiratory (e.g., empyema, lung abscess), cardiac (e.g.,infective endocarditis), gastrointestinal (e.g., secretory diarrhoea,splenic absces, retroperitoneal abscess), CNS (e.g., cerebral abscess),eye (e.g., blepharitis, conjunctivitis, lkeratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric absces, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis).

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Altemnatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized”; where thecomplimentarily determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody , for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal.,(1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS, 1986:83,9551), encapsulation of DNA in various forms ofliposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS 1984:81,5849).

Antagonists and Agonists—Assays and Molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. TIhesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1 (2). Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of ratBpolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bacteriocidal. The method of screening may involvehigh-thoughput techniques. For example, to screen for agonists orantagoists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising ratB polypeptide and/or ratC polypeptide [SEQ ID NO:6] and/orratA polypeptide [SEQ ID NO:8] and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a ratB agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the ratB polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of ratB polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in ratB polynucleotide or polypeptide activity,and binding assays known in the art.

TABLE 2 Polynucleotide sequence of rat C [SEQ ID NO:5] 5′-ATGACAAAAGTAACACGTGAAGAAGTTGAGCATATCGCGAATCTTGCAAGACTTCAAATTTCTCCTGAAGAAACGGAAGAAATGGCCAACACATTAGAAAGCATTTTAGATTTTGCAAAACAAAATGATAGCGCTGATACAGAAGGCGTTGAACCTACATATCACGTTTTAGATTTACAAAACGTTTTACGTGAAGATAAAGCAATTAAAGGTATTCCGCAAGAATTAGCTTTGAAAAATGCCAAAGAAACAGAAGATGGACAATTTAAAGTGCCTACAATCATGAATGAGGAGGACGCG-3′ Polypeptide sequence of rat C [SEQ ID NO:6]deduced from the sequence of SEQ ID NO:3. NH₂-MTKVTREEVEHIANLARLQISPEETEEMANTLESILDFAKQNDSADTEGVEPTYHVLDLQNVLREDKAIKGIPQELALKNAKETEDGQFKVPTIMNEEDA-COOH Polynucleotide sequence of ratA [SEQID NO:7] 5′-ATGAGCATTCGCTACGAATCGGTTGAGAATTTATTAACTTTAATAAAAGACAAAAAAATCAAACCATCTGATGTTGTTAAAGATATATATGATGCAATTGAAGAGACTGATCCAACAATTAAGTCTTTTCTAGCGCTGGATAAAGAAAATGCAATCAAAAAAGCGCAAGAATTGGATGAATTACAAGCAAAAGATCAAATGGATGGCAAATTATTTGGTATTCCAATGGGTATAAAAGATAACATTATTACAAACGGATTAGAAACAACATGTGCAAGTAAAATGTTAGAAGGTTTTGTGCCAATTTACGAATCTACTGTAATGGAAAAACTACATAAAGAGAATGCCGTTTTAATCGGTAAATTAAATATGGATGAGTTTGCAATGGGTGGTTCAACAGAAACATCTTATTTCAAAAAAACAGTTAACCCATTTGACCATAAAGCAGTACCAGGTGGTTCATCAGGTGGATCTGCAGCAGCAGTTGCAGCTGGCTTAGTACCATTTAGCTTAGGTTCAGACACAGGTGGTTCAATTAGACAACCGGCTGCATATTGTGGCGTTGTCGGTATGAAACCAACATACGGTCGTGTATCTCGATTTGGATTAGTTGCTTTTGCATCTTCATTAGACCAAATTGGTCCATTGACTCGAAATGTAAAAGATAATGCAATCGTATTAGAAGCTATTTCTGGTGCAGATGTTAATGACTCTACAAGTGCACCAGTTGATGATGTAGACTTTACATCTGAAATTGGTAAAGATATTAAAGGATTAAAAGTTGCATTACCTAAAGAATACTTAGGTGAAGGTGTAGCTGATGACGTAAAAGAAGCAGTTCAAAACGCTGTAGAAACTTTAAAATCTTTAGGTGCTGTCGTTGAGGAAGTATCATTGCCAAATACTAAATTTGGTATTCCATCATATTACGTGATTGCATCATCAGAAGCTTCGTCAAACCTTTCTCGTTTTGACGGAATTCGTTATGGTTATCATTCTAAAGAAGCTCATTCATTAGAAGAATTATATAAAATGTCAAGATCTGAAGGTTTCGGTAAAGAAGTAAAACGTCGTATTTTCTTAGGTACATTTGCATTAAGTTCAGGTTACTACGATGCTTACTATAAAAAATCTCAAAAAGTTAGAACATTGATTAAAAATGACTTTGATAAAGTATTCGAAAATTATGATGTAGTAGTTGGTCCAACAGCGCCTACAACTGCGTTTAATTTAGGTGAAGAAATTGATGATCCATTAACAATGTATGCCAATGATTTATTAACAACACCAGTAAACTTAGCTGGATTACCTGGTATTTCTGTTCCTTGTGGACAATCAAATGGCCGACCAATCGGTTTACAGTTCATTGGTAAACCATTCGATGAAAAAACGTTATATCGTGTCGCTTATCAATATGAAACACAATACAATTTACATGACGTTTATGAAAAATTA-3′ Polypetidesequence of ratA [SEQ ID NO:8] deduced from the sequence of SEQ ID NO:5.NH₂-MSIRYESVENLLTLIKDKKIKPSDVVKDIYDAIEETDPTIKSFLALDKENAIKKAQELDELQAKDQMDGKLFGIPMGIKDNIITNGLETTCASKMLEGFVPIYESTVMEKLHKENAVLIGKLNMDEFAMGGSTETSYFKKTVNPFDHKAVPGGSSGGSAAAVAAGLVPFSLGSDTGGSIRQPAAYCGVVGMKPTYGRVSRFGLVAFASSLDQIGPLTRNVKDNAIVLEAISGADVNDSTSAPVDDVDFTSEIGKDIKGLKVALPKEYLGEGVADDVKEAVQNAVETLKSLGAVVEEVSLPNTKFGIPSYYVIASSEASSNLSRFDGIRYGYHSKEAHSLEELYKMSRSEGFGKEVKRRIFLGTFALSSGYYDAYYKKSQKVRTLIKNDFDKVFENYDVVVGPTAPTTAFNLGEEIDDPLTMYANDLLTTPVNLAGLPGISVPCGQSNGRPIGLQFIGKPFDEKTLYRVAYQYETQYNLHDVYEKL-COOH

Another example of an assay for ratB antagonists is a competitive assaythat combines ratB and/or ratC [SEQ ID NO:5 or 6] and/or ratA [SEQ IDNO:7 or 8] and a potential antagonist with ratB-binding molecules,recombinant ratB binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. ratB can bc labeled, such as byradioactivity or a colorimetric compound, such that the number of ratBmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polyiucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing ratB-induced activities, thereby preventing the action of ratBby excluding ratB polypeptide and/or ratC polypeptide [SEQ ID NO:6]and/or ratA polypeptide [SEQ ID NO:8] from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, pcptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of ratB.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino teininalregions of the encoded protein or Shine-Delgamo or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock ratB protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal, Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial ratB proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat disease, such as, infections of the upperrespiratoly tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretoly diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darciyocystitis), kidney and urinary tract (e.g., epididymitis,int-arenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with ratB, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Staphylococcus aureus infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector to direct expression of ratB, or afragment or a variant thereof, for expressing ratB, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as to produce antibody and/or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not. One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise.

Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid,or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a ratB or protein coded therefrom,wherein the composition comprises a recombinant ratB or protein codedtherefrom comprising DNA which codes tor and expresses an antigen ofsaid ratB or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+ T cells.

A ratB polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an anti genic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintrader-mal. Fonrulations suitable for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe fonnulation insotonic with the bodily fluid, preferably the blood,of the individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain ratBprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, Kits and Administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agyonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or calTiers for use with cells, tissuesor organisms, such as a phanraceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the fonin of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formiulations may alsocontain compatible conventional calTiers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute Up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinalycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

Many orthopacdic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable fone. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carTied out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain Selection, Library Production and Sequencing

The polynucleotide having the DNA secquence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Staphylococcusaureus in E. coli. The sequencing data from two or more clonescontaining overlapping Staphylococcus aureus DNAs was used to constructthe contiguous DNA sequence in SEQ ID NO:1. Libraries may be prepared byroutine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E.coli infected with the packagedlibrary. The library is amplified by standard procedures.

8 1425 base pairs nucleic acid double linear unknown 1 ATGCATTTTGAAACAGTTAT AGGACTTGAA GTTCACGTAG AGTTAAAAAC GGACTCAAAA 60 ATGTTTTCTCCATCACCAGC GCATTTTGGA GCAGAACCTA ACTCAAATAC AAATGTTATC 120 GACTTAGCATATCCAGGTGT CTTACCAGTT GTTAATAAGC GTGCAGTAGA CTGGGCAATG 180 CGTGCTGCAATGGCACTAAA TATGGAAATC GCAACAGAAT CTAAGTTTGA CCGTAAGAAC 240 TATTTCTATCCAGATAATCC AAAAGCATAT CAAATTTCTC AATTTGATCA ACCAATTGGT 300 GAAAATGGATATATCGATAT CGAAGTCGAC GGTGAAACAA AACGAATCGG TATTACTCGT 360 CTTCACATGGAAGAAGATGC TGGTAAGTCA ACACATAAAG GTGAGTATTC ATTAGTTGAC 420 TTGAACCGTCAAGGTACACC GCTAATTGAA ATCGTATCTG AACCAGATAT TCGTTCACCT 480 AAAGAAGCATATGCATATTT AGAAAAATTA CGTTCAATTA TTCAATACAC TGGTGTATCA 540 GACGTTAAGATGGAAGAGGG ATCTTTACGT TGTGATGCTA ACATCTCTTT GCGTCCATAT 600 GGTCAAGAAAAATTTGGTAC TAAAGCCGAA TTGAAAAACT TAAACTCATT TAACTATGTA 660 CGTAAAGGTTTAGAATATGA AGAAAAACGC CAAGAAGAAG AATTGTTAAA TGGTGGAGAA 720 ATCGGACAAGAAACACGTCG ATTTGATGAA TCTACAGGTA AAACAATTTT AATGCGTGTT 780 AAAGAAGGTTCTGATGATTA CCGTTACTTC CCAGAGCCTG ACATTGTACC TTTATATATT 840 GATGATGCTTGGAAAGAGCG TGTTCGTCAG ACAATTCCTG AATTACCAGA TGAGCGTAAG 900 GCTAAGTATGTAAATGAATT AGGTTTACCT GCATACGATG CACACGTATT AACATTGACT 960 AAAGAAATGTCAGATTTCTT TGAATCAACA ATTGAACACG GTGCAGATGT TAAATTAACA 1020 TCTAACTGGTTAATGGGTGG CGTAAACGAA TATTTAAATA AAAATCAAGT AGAATTATTA 1080 GATACTAAATTAACACCAGA AAATTTAGCA GGTATGATTA AACTTATCGA AGACGGAACA 1140 ATGAGCAGTAAAATTGCGAA GAAAGTCTTC CCAGAGTTAG CAGCTAAAGG TGGTAATGCT 1200 AAACAGATTATGGAAGATAA TGGCTTAGTT CAAATTTCTG ATGAAGCAAC ACTTCTAAAA 1260 TTTGTAAATGAAGCATTAGA CAATAACGAA CAATCAGTTG AAGATTACAA AAATGGTAAA 1320 GGCAAAGCTATGGGCTTCTT AGTTGGTCAA ATTATGAAAG CGTCTAAAGG TCAAGCTAAT 1380 CCACAATTAGTAAATCAACT ATTAAAACAA GAATTAGATA AAAGA 1425 475 amino acids amino acidsingle linear unknown 2 Met His Phe Glu Thr Val Ile Gly Leu Glu Val HisVal Glu Leu Lys 1 5 10 15 Thr Asp Ser Lys Met Phe Ser Pro Ser Pro AlaHis Phe Gly Ala Glu 20 25 30 Pro Asn Ser Asn Thr Asn Val Ile Asp Leu AlaTyr Pro Gly Val Leu 35 40 45 Pro Val Val Asn Lys Arg Ala Val Asp Trp AlaMet Arg Ala Ala Met 50 55 60 Ala Leu Asn Met Glu Ile Ala Thr Glu Ser LysPhe Asp Arg Lys Asn 65 70 75 80 Tyr Phe Tyr Pro Asp Asn Pro Lys Ala TyrGln Ile Ser Gln Phe Asp 85 90 95 Gln Pro Ile Gly Glu Asn Gly Tyr Ile AspIle Glu Val Asp Gly Glu 100 105 110 Thr Lys Arg Ile Gly Ile Thr Arg LeuHis Met Glu Glu Asp Ala Gly 115 120 125 Lys Ser Thr His Lys Gly Glu TyrSer Leu Val Asp Leu Asn Arg Gln 130 135 140 Gly Thr Pro Leu Ile Glu IleVal Ser Glu Pro Asp Ile Arg Ser Pro 145 150 155 160 Lys Glu Ala Tyr AlaTyr Leu Glu Lys Leu Arg Ser Ile Ile Gln Tyr 165 170 175 Thr Gly Val SerAsp Val Lys Met Glu Glu Gly Ser Leu Arg Cys Asp 180 185 190 Ala Asn IleSer Leu Arg Pro Tyr Gly Gln Glu Lys Phe Gly Thr Lys 195 200 205 Ala GluLeu Lys Asn Leu Asn Ser Phe Asn Tyr Val Arg Lys Gly Leu 210 215 220 GluTyr Glu Glu Lys Arg Gln Glu Glu Glu Leu Leu Asn Gly Gly Glu 225 230 235240 Ile Gly Gln Glu Thr Arg Arg Phe Asp Glu Ser Thr Gly Lys Thr Ile 245250 255 Leu Met Arg Val Lys Glu Gly Ser Asp Asp Tyr Arg Tyr Phe Pro Glu260 265 270 Pro Asp Ile Val Pro Leu Tyr Ile Asp Asp Ala Trp Lys Glu ArgVal 275 280 285 Arg Gln Thr Ile Pro Glu Leu Pro Asp Glu Arg Lys Ala LysTyr Val 290 295 300 Asn Glu Leu Gly Leu Pro Ala Tyr Asp Ala His Val LeuThr Leu Thr 305 310 315 320 Lys Glu Met Ser Asp Phe Phe Glu Ser Thr IleGlu His Gly Ala Asp 325 330 335 Val Lys Leu Thr Ser Asn Trp Leu Met GlyGly Val Asn Glu Tyr Leu 340 345 350 Asn Lys Asn Gln Val Glu Leu Leu AspThr Lys Leu Thr Pro Glu Asn 355 360 365 Leu Ala Gly Met Ile Lys Leu IleGlu Asp Gly Thr Met Ser Ser Lys 370 375 380 Ile Ala Lys Lys Val Phe ProGlu Leu Ala Ala Lys Gly Gly Asn Ala 385 390 395 400 Lys Gln Ile Met GluAsp Asn Gly Leu Val Gln Ile Ser Asp Glu Ala 405 410 415 Thr Leu Leu LysPhe Val Asn Glu Ala Leu Asp Asn Asn Glu Gln Ser 420 425 430 Val Glu AspTyr Lys Asn Gly Lys Gly Lys Ala Met Gly Phe Leu Val 435 440 445 Gly GlnIle Met Lys Ala Ser Lys Gly Gln Ala Asn Pro Gln Leu Val 450 455 460 AsnGln Leu Leu Lys Gln Glu Leu Asp Lys Arg 465 470 475 2201 base pairsnucleic acid double linear unknown 3 CTGAAAAACT TAAACTCATT TAACTATGTACGTAAAGGTT TAGAATATGA AGAAAAACGC 60 CAAGAAGAAG AATTGTTAAA TGGTGGAGAAATCGGACAAG AAACACGTCG ATTTGATGAA 120 TCTACAGGTA AAACAATTTT AATGCGTGTTAAAGAAGGTT CTGATGATTA CCGNTACTTC 180 CCAGAGCCTG ACATTGTACC TTTATATATTGATGATGCTT GGAAAGAGCG TGTTCGTCAG 240 ACAATTCCTG AATTACCAGA TGAGCGTAAGGCTAAGTATG TAAATGAATT AGGTTTACTG 300 CATACGATGC NCNCGTATTA ACNTTGACTAAAGAAATGTC AGATTTCTTT GAATCAACAA 360 TTGGAACNAG GGTGCAGATT GTTAAATTTAACATCTAACT GGGTTAATGG GTGGCGTNAA 420 CGAATATTTA AATAAAAATC AANTAGAATTATTAGATACT AAATTAACAC CAGAAAATTT 480 AGCAGGTATG ATTAAACTTA TCGAAGACGGAACAATGAGC AGTAAAATTG CGAAGAAAGT 540 CTTCCCAGAG TTAGGCAGCT AAAGGGTGGGTAATGCTAAA CAGATTATGG GANGATAATG 600 GCTNAGTTCA AATTTCTTGA TGGAAGCAACAATCTTCTAA AATTGGGTAN ATGGAAGCAT 660 TAGACAAATA ACGAACAATC NGTGGAAGATTACAAAAATG GTAAAGGCAA AGCTATGGGG 720 CTTCTTAGTT GGTCAAATTA TGAAAGCGTCTAAAGGTCAA GCTAATCCAC AATTAGTAAA 780 TCAACTATTA AAACAAGAAT TAGATAAAAGATAATTTANA TCATCAAACT ATGAAGATTT 840 AAAAAATAAA CCCTTGATTG CTGACTTAGATGCAATCGAG GGTTTATTTA TATCTATAGA 900 AGTCATATTA CTTTTAACTT TATTCATTGNACATGTTAAT GGTAAAAATA TTAATTTTAT 960 TAATGCGTTA GCTTTAATTA TATTAAGGCAAACTGTATAA TAAAAAGGTA TAAAACATTT 1020 GTGTATAAAG ACAACATTAT ATTTACAACATCATTTTAAA GGTAAAATAG CATAACTGAC 1080 GAAGTCTATA TAATGAAGAA CGGCAAAAAATGCTGAATAA ATAACAAGCT TTGTACATAT 1140 TGAGATAGTA TTTGTTTAAG ATACAAGTTGGTCTTTAACG ATATTAAGAA TGATGAAATA 1200 AGACTGAGCC TGGGTCATAA ATTCAATGTCCTAGGCACTA CAATGTTAAT ATTGGCAGTA 1260 GTTGACTGAA AGAAAATACG CTTGTAACAAGCTTNNNTCA ATTCTAGTGG GGCCCCAACA 1320 TAGAAGCTGA CTTTCTGTCA GCTTACAATAATGTGCAAGT NGGGGTGGGG CCCCAAACAA 1380 AGAGAATTTC GAAAGGAAAT TCTACAGACAATGCAAGTTG GGGTAGAACG AAATAAATTT 1440 TGTTAAATAT TATTTCTGTC CCACTCCCTATTAGACGAAA CAAAGATGAA GTCAAAATAT 1500 ATGAATTTTA AGTAGAAGGA TAAGATATGAACAAACGTGC TAGAATCATT TATAACCCGA 1560 CATCAGGTAA AGAGCTAATT TAAAAGAGAATTACCTGATG CCTTAATAAA ATTAGAAAAA 1620 GCGGGATATG AAACGAGTGC ATATGCAACCGAGAAAATAG GTGATGCCAC ACTTGAAGCA 1680 GAAAGAGCTA TGCATGAAAA TTATGATGTATTAATCGCTG CAGGTGGTGA TGGAACATTA 1740 AATGAAGTAG TTAATGGTAT CGCAGAAAAGCCTAATCGTC CTAAGCTAGG TGTCATTCCT 1800 ATGGGTACTG TTAATGACTT TGGACGTGCATTGCATATAC CTAATGACAT CATGGGGGCA 1860 CTTGATGTCA TCATTGAAGG TCATTCTACTAAAGTAGATA TTGGTAAAAT GAATAATCGA 1920 TACTTTATTA ATTTAGCTGC AGGCGGACAATTGACGCAAG TCTCTTATGA AACACCGAGT 1980 AAATTGAAAT CTATTGTTGG TCCATTTGCTTATTACATCA AAGGTTTCGA AATGTTACCT 2040 CAAATGAAAG CTGTAGATTT AAGAATTGAATATGATGGTA ATGTTTNCCA AGGAGAAGCA 2100 TTATTATTCT TTNTAGGCTT AACAAATCCAATGGCAGGAT TCGAAAAATT AGTGCCCGGA 2160 CGCTAAGTTA GATGACGGCT ATTTCTACGTTNAATNTATA G 2201 106 amino acids amino acid single linear unknown 4 MetLys Asn Leu Asn Ser Phe Asn Tyr Val Arg Lys Gly Leu Glu Tyr 1 5 10 15Glu Glu Lys Arg Gln Glu Glu Glu Leu Leu Asn Gly Gly Glu Ile Gly 20 25 30Gln Glu Thr Arg Arg Phe Asp Glu Ser Thr Gly Lys Thr Ile Leu Met 35 40 45Arg Val Lys Glu Gly Ser Asp Asp Tyr Arg Tyr Phe Pro Glu Pro Asp 50 55 60Ile Val Pro Leu Tyr Ile Asp Asp Ala Trp Lys Glu Arg Val Arg Gln 65 70 7580 Thr Ile Pro Glu Leu Pro Asp Glu Arg Lys Ala Lys Tyr Val Asn Glu 85 9095 Leu Gly Leu Leu His Thr Met Xaa Xaa Tyr 100 105 300 base pairsnucleic acid double linear unknown 5 ATGACAAAAG TAACACGTGA AGAAGTTGAGCATATCGCGA ATCTTGCAAG ACTTCAAATT 60 TCTCCTGAAG AAACGGAAGA AATGGCCAACACATTAGAAA GCATTTTAGA TTTTGCAAAA 120 CAAAATGATA GCGCTGATAC AGAAGGCGTTGAACCTACAT ATCACGTTTT AGATTTACAA 180 AACGTTTTAC GTGAAGATAA AGCAATTAAAGGTATTCCGC AAGAATTAGC TTTGAAAAAT 240 GCCAAAGAAA CAGAAGATGG ACAATTTAAAGTGCCTACAA TCATGAATGA GGAGGACGCG 300 100 amino acids amino acid singlelinear unknown 6 Met Thr Lys Val Thr Arg Glu Glu Val Glu His Ile Ala AsnLeu Ala 1 5 10 15 Arg Leu Gln Ile Ser Pro Glu Glu Thr Glu Glu Met AlaAsn Thr Leu 20 25 30 Glu Ser Ile Leu Asp Phe Ala Lys Gln Asn Asp Ser AlaAsp Thr Glu 35 40 45 Gly Val Glu Pro Thr Tyr His Val Leu Asp Leu Gln AsnVal Leu Arg 50 55 60 Glu Asp Lys Ala Ile Lys Gly Ile Pro Gln Glu Leu AlaLeu Lys Asn 65 70 75 80 Ala Lys Glu Thr Glu Asp Gly Gln Phe Lys Val ProThr Ile Met Asn 85 90 95 Glu Glu Asp Ala 100 1455 base pairs nucleicacid double linear unknown 7 ATGAGCATTC GCTACGAATC GGTTGAGAAT TTATTAACTTTAATAAAAGA CAAAAAAATC 60 AAACCATCTG ATGTTGTTAA AGATATATAT GATGCAATTGAAGAGACTGA TCCAACAATT 120 AAGTCTTTTC TAGCGCTGGA TAAAGAAAAT GCAATCAAAAAAGCGCAAGA ATTGGATGAA 180 TTACAAGCAA AAGATCAAAT GGATGGCAAA TTATTTGGTATTCCAATGGG TATAAAAGAT 240 AACATTATTA CAAACGGATT AGAAACAACA TGTGCAAGTAAAATGTTAGA AGGTTTTGTG 300 CCAATTTACG AATCTACTGT AATGGAAAAA CTACATAAAGAGAATGCCGT TTTAATCGGT 360 AAATTAAATA TGGATGAGTT TGCAATGGGT GGTTCAACAGAAACATCTTA TTTCAAAAAA 420 ACAGTTAACC CATTTGACCA TAAAGCAGTA CCAGGTGGTTCATCAGGTGG ATCTGCAGCA 480 GCAGTTGCAG CTGGCTTAGT ACCATTTAGC TTAGGTTCAGACACAGGTGG TTCAATTAGA 540 CAACCGGCTG CATATTGTGG CGTTGTCGGT ATGAAACCAACATACGGTCG TGTATCTCGA 600 TTTGGATTAG TTGCTTTTGC ATCTTCATTA GACCAAATTGGTCCATTGAC TCGAAATGTA 660 AAAGATAATG CAATCGTATT AGAAGCTATT TCTGGTGCAGATGTTAATGA CTCTACAAGT 720 GCACCAGTTG ATGATGTAGA CTTTACATCT GAAATTGGTAAAGATATTAA AGGATTAAAA 780 GTTGCATTAC CTAAAGAATA CTTAGGTGAA GGTGTAGCTGATGACGTAAA AGAAGCAGTT 840 CAAAACGCTG TAGAAACTTT AAAATCTTTA GGTGCTGTCGTTGAGGAAGT ATCATTGCCA 900 AATACTAAAT TTGGTATTCC ATCATATTAC GTGATTGCATCATCAGAAGC TTCGTCAAAC 960 CTTTCTCGTT TTGACGGAAT TCGTTATGGT TATCATTCTAAAGAAGCTCA TTCATTAGAA 1020 GAATTATATA AAATGTCAAG ATCTGAAGGT TTCGGTAAAGAAGTAAAACG TCGTATTTTC 1080 TTAGGTACAT TTGCATTAAG TTCAGGTTAC TACGATGCTTACTATAAAAA ATCTCAAAAA 1140 GTTAGAACAT TGATTAAAAA TGACTTTGAT AAAGTATTCGAAAATTATGA TGTAGTAGTT 1200 GGTCCAACAG CGCCTACAAC TGCGTTTAAT TTAGGTGAAGAAATTGATGA TCCATTAACA 1260 ATGTATGCCA ATGATTTATT AACAACACCA GTAAACTTAGCTGGATTACC TGGTATTTCT 1320 GTTCCTTGTG GACAATCAAA TGGCCGACCA ATCGGTTTACAGTTCATTGG TAAACCATTC 1380 GATGAAAAAA CGTTATATCG TGTCGCTTAT CAATATGAAACACAATACAA TTTACATGAC 1440 GTTTATGAAA AATTA 1455 485 amino acids aminoacid single linear unknown 8 Met Ser Ile Arg Tyr Glu Ser Val Glu Asn LeuLeu Thr Leu Ile Lys 1 5 10 15 Asp Lys Lys Ile Lys Pro Ser Asp Val ValLys Asp Ile Tyr Asp Ala 20 25 30 Ile Glu Glu Thr Asp Pro Thr Ile Lys SerPhe Leu Ala Leu Asp Lys 35 40 45 Glu Asn Ala Ile Lys Lys Ala Gln Glu LeuAsp Glu Leu Gln Ala Lys 50 55 60 Asp Gln Met Asp Gly Lys Leu Phe Gly IlePro Met Gly Ile Lys Asp 65 70 75 80 Asn Ile Ile Thr Asn Gly Leu Glu ThrThr Cys Ala Ser Lys Met Leu 85 90 95 Glu Gly Phe Val Pro Ile Tyr Glu SerThr Val Met Glu Lys Leu His 100 105 110 Lys Glu Asn Ala Val Leu Ile GlyLys Leu Asn Met Asp Glu Phe Ala 115 120 125 Met Gly Gly Ser Thr Glu ThrSer Tyr Phe Lys Lys Thr Val Asn Pro 130 135 140 Phe Asp His Lys Ala ValPro Gly Gly Ser Ser Gly Gly Ser Ala Ala 145 150 155 160 Ala Val Ala AlaGly Leu Val Pro Phe Ser Leu Gly Ser Asp Thr Gly 165 170 175 Gly Ser IleArg Gln Pro Ala Ala Tyr Cys Gly Val Val Gly Met Lys 180 185 190 Pro ThrTyr Gly Arg Val Ser Arg Phe Gly Leu Val Ala Phe Ala Ser 195 200 205 SerLeu Asp Gln Ile Gly Pro Leu Thr Arg Asn Val Lys Asp Asn Ala 210 215 220Ile Val Leu Glu Ala Ile Ser Gly Ala Asp Val Asn Asp Ser Thr Ser 225 230235 240 Ala Pro Val Asp Asp Val Asp Phe Thr Ser Glu Ile Gly Lys Asp Ile245 250 255 Lys Gly Leu Lys Val Ala Leu Pro Lys Glu Tyr Leu Gly Glu GlyVal 260 265 270 Ala Asp Asp Val Lys Glu Ala Val Gln Asn Ala Val Glu ThrLeu Lys 275 280 285 Ser Leu Gly Ala Val Val Glu Glu Val Ser Leu Pro AsnThr Lys Phe 290 295 300 Gly Ile Pro Ser Tyr Tyr Val Ile Ala Ser Ser GluAla Ser Ser Asn 305 310 315 320 Leu Ser Arg Phe Asp Gly Ile Arg Tyr GlyTyr His Ser Lys Glu Ala 325 330 335 His Ser Leu Glu Glu Leu Tyr Lys MetSer Arg Ser Glu Gly Phe Gly 340 345 350 Lys Glu Val Lys Arg Arg Ile PheLeu Gly Thr Phe Ala Leu Ser Ser 355 360 365 Gly Tyr Tyr Asp Ala Tyr TyrLys Lys Ser Gln Lys Val Arg Thr Leu 370 375 380 Ile Lys Asn Asp Phe AspLys Val Phe Glu Asn Tyr Asp Val Val Val 385 390 395 400 Gly Pro Thr AlaPro Thr Thr Ala Phe Asn Leu Gly Glu Glu Ile Asp 405 410 415 Asp Pro LeuThr Met Tyr Ala Asn Asp Leu Leu Thr Thr Pro Val Asn 420 425 430 Leu AlaGly Leu Pro Gly Ile Ser Val Pro Cys Gly Gln Ser Asn Gly 435 440 445 ArgPro Ile Gly Leu Gln Phe Ile Gly Lys Pro Phe Asp Glu Lys Thr 450 455 460Leu Tyr Arg Val Ala Tyr Gln Tyr Glu Thr Gln Tyr Asn Leu His Asp 465 470475 480 Val Tyr Glu Lys Leu 485

What is claimed is:
 1. An isolated polynucleotide segment comprising a first polynucleotide sequence or the full complement of the entire length of the first polynucleotide sequence, wherein the first polynucleotide sequence is identical to SEQ ID NO:1, except that, over the entire length corresponding to SEQ ID NO:1, n_(n) nucleotides are substituted, inserted or deleted, wherein n_(n) satisfies the following expression n _(n) ≦x _(n)−(x _(n) •y) wherein x_(n) is the total number of nucleotides in SEQ ID NO:1, y is at least 0.95, and wherein any non-integer product of x_(n) and y is rounded down to the nearest integer before subtracting the product from x_(n).
 2. A vector comprising the isolated polynucleotide segment of claim
 1. 3. An isolated host cell comprising the vector of claim
 2. 4. The isolated polynucleotide segment of claim 1, wherein y is at least 0.97.
 5. The isolated polynucleotide segment of claim 1, wherein y is at least 0.99.
 6. The isolated polynucleotide segment of claim 1, wherein the first polynucleotide sequence comprises SEQ ID NO:1.
 7. A polynucleotide which encodes a fusion polypeptide and which includes the isolated polynucleotide segment of claim
 6. 8. A vector comprising the isolated polynucleotide segment of claim
 6. 9. An isolated host cell comprising the vector of claim
 8. 10. A process for producing a polypeptide comprising the step of culturing the host cell of claim 9 under conditions sufficient for the production of the polypeptide, wherein the polypeptide is encoded by the first polynucleotide sequence.
 11. An isolated polynucleotide segment, comprising a first polynucleotide sequence or the full complement of the entire length of the first polynucleotide sequence, wherein the first polynucleotide sequence encodes the same polypeptide, expressed by the ratB gene encoded by a nucleic acid sequence comprising SEQ ID NO:1 contained in Staphylococcus aureus contained in NCIMB Deposit No.
 40771. 12. A polynucleotide which encodes a fusion polypeptide and which includes the isolated polynucleotide segment of claim
 11. 13. An isolated polynucleotide segment, comprising a first polyntucleotide sequence or the full complement of the entire length of the first polynucleotide sequence, wherein the first polynucleotide sequence hybridizes to the full complement of SEQ ID NO:1, wherein the hybridization conditions include incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing in 0.1×SSC at 65° C.; and, wherein the first polynucleotide sequence is identical to SEQ ID NO:1, except that, over the entire length corresponding to SEQ ID NO:1, n_(n) nucleotides are substituted, inserted or deleted, wherein n_(n) satisfies the following expression n _(n) ≦x _(n)−(x _(n) •y) wherein x_(n) is the total number of nucleotides in SEQ ID NO:1, y is at least 0.95, and wherein any non-integer product of x_(n) and y is rounded down to the nearest integer before subtracting the product from x_(n).
 14. The isolated polynucleotide segment of claim 13, wherein y is at least 0.97.
 15. An isolated polynucleotide segment comprising a first polynucleotide sequence or the full complement of the entire length of the first polynucleotide sequence, wherein the first polynucleotide sequence encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2.
 16. A vector comprising the isolated polynucleotide segment of claim
 15. 17. An isolated host cell comprising the vector of claim
 16. 18. A process for producing a polypeptide comprising the step of culturing the host cell of claim 17 under conditions sufficient for the production of the polypeptide, wherein the polypeptide is encoded by the first polynucleotide sequence.
 19. A polynucleotide which encodes a fusion polypeptide and which includes the isolated polynucleotide segment of claim
 15. 20. An isolated polynucleotide segment comprising a first polynucleotide sequence or the full complement of the entire length of the first polynucleotide sequence, wherein the first polynucleotide sequence encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2.
 21. A vector comprising the isolated polynucleotide segment of claim
 20. 22. An isolated host cell comprising the vector of claim
 21. 23. A process for producing a polypeptide comprising the step of culturing the host cell of claim 22 under conditions sufficient for the production of the polypeptide, wherein the polypeptide is encoded by the first polynucleotide sequence. 